KR102075160B1 - Mutant strain with enhanced L-glutamic acid productivity and method for preparing L-glutamic acid using the same - Google Patents

Abstract

The present invention relates to a mutant strain with enhanced L-glutamic acid productivity, and a method for preparing L-glutamic acid using the same. The mutant strain according to an embodiment of the present invention weakens or inactivates the activity of citramalate synthase, thereby reducing the production amount of by-products, citramalate, and having excellent L-glutamic acid productivity. In addition, the strain with a changed YggB protein enhances the release of glutamic acid, thereby improving the production yield of L-glutamic acid. Therefore, L-glutamic acid can be more effectively produced by using the mutant strain.

Description

Mutant strain with enhanced L-glutamic acid productivity and method for preparing L-glutamic acid using the same}

The present invention relates to a mutant strain having improved L-glutamic acid production capacity and a method for producing L-glutamic acid using the same, and more specifically, citramalate as a byproduct due to a weakened or inactivated activity of citramalate synthase. The present invention relates to a Corynebacterium sp. Mutant strain having a reduced production amount and an increased production capacity of L-glutamic acid, and a method for producing L-glutamic acid using the same.

L-glutamic acid is a representative amino acid produced by microbial fermentation. Sodium L-glutamate (MSG) balances the overall taste of food, allowing meat, fish, chicken, vegetables, sauces, soups, It is widely used as seasoning for home and processed food production because it can improve the taste of foods such as seasoning and improve the taste of low salt foods with reduced salt by 30%.

In order to produce L-glutamic acid, it is usually produced through fermentation mainly using strains of the genus Brevibacterium or Corynebacterium and its variants. In order to increase the production of L-glutamic acid by microbial culture, in order to increase the expression of the gene in the L-glutamic acid biosynthesis pathway, the number of copies of the gene is increased or the promoter of the gene is modified to regulate the enzyme activity on the biosynthetic pathway. The method has been used. Specifically, amplification of specific genes such as the pyc gene or fasR (US Pat. No. 6,852,516) or the promoter regions of the gdh, gltA, icd , pdh and argG genes (US Pat. No. 6,962,805) L- Glutamic acid production has been increased.

Citramalate synthase ( cimA ) is an enzyme that synthesizes citramalate by using acetyl-CoA and pyruvate as a substrate, and there is no cimA gene in Corynebacterium glutamicum and corynebacterium. In glutamicum, the leuA gene, which is highly homologous to the cimA gene, is known to play a role of citramalate synthase.

Since acetyl-CoA is used as a substrate in the fermentation route of L-glutamic acid, the inventors of the present application attenuate or inactivate the activity of citramalate synthase to save the substrate acetyl-CoA to further increase L-glutamic acid productivity. Now.

Meanwhile, the yggB gene of coryneform bacteria is an analog of the yggB gene of Escherichia coli (FEMS Microbiol Lett. 2003, 218 (2), p305-309, Mol Microbiol. 2004, 54 (2), p420-438) It has been reported to be a type of mechanosensitive channel (EMBO J. 1999, 18 (7): 1730-7). As a method of imparting L-glutamic acid producing ability to coryneform bacteria, specifically, the effect of a variation such as the present invention on L-glutamic acid production has not been reported.

Therefore, the inventors of the present application induce a mutation in a mutant strain that attenuates or inactivates the activity of citramalate synthase, and additionally, YggB protein involved in glutamic acid excretion, thereby producing a mutant strain, and the mutant strain has an ability to produce L-glutamic acid. It confirmed that it was excellent and completed this invention.

Republic of Korea Patent No. 10-1138289

It is an object of the present invention to provide a Corynebacterium sp. Variant strain in which the activity of citramalate synthase is attenuated or inactivated and L-glutamic acid is produced.

Another object of the present invention

(a) culturing a strain of genus Corynebacterium in a medium; And

(b) recovering L-glutamic acid from the cultured strain or culture medium to provide a method for producing L-glutamic acid.

One aspect of the present invention provides a Corynebacterium sp. Mutant strain in which the activity of citramalate synthase is attenuated or inactivated and L-glutamic acid is produced.

In the present invention, "citramalate synthase" refers to an enzyme that catalyzes the process of converting acetyl-CoA and pyruvate to citramalate and coenzyme A.

In the present invention, the "enhanced production" strain means that the productivity of L-glutamic acid is increased compared to the parent strain.

In the present invention, "parent strain" refers to a wild-type or variant strain to be subjected to the mutation, and includes a subject that is directly subjected to the mutation or transformed with a recombinant vector or the like. In the present invention, the parent strain may be a wild type Corynebacterium glutamicum strain or a strain mutated from the wild type. Preferably, it may be Corynebacterium glutamicum KCTC 11558BP strain.

According to one embodiment of the present invention, the Corynebacterium sp. Mutant strain having improved production capacity of L-glutamic acid has a weakened or inactivated activity of citramalate synthase compared to the parent strain. It may be.

In the present invention, "weakened activity" means a case where the degree of citramalate synthase enzyme activity in the cell is reduced compared to the parent strain, such as a wild type strain or a strain before modification. The weakening is due to a mutation of the gene encoding the enzyme, and the activity of the enzyme itself is reduced compared to the activity of the enzyme originally possessed by the microorganism, and the inhibition of transcription or translation of the gene encoding the enzyme. When the expression level of the enzyme is lowered and the overall level of enzymatic activity in the cell is lower than that of the wild type strain or the strain before modification, the concept also includes a combination thereof. In addition, in the present invention, "inactivation" refers to a state in which the expression of the gene encoding citramalate synthase is not expressed at all, or inactive even if expressed, compared to the wild type strain or the strain before modification.

According to one embodiment of the present invention, attenuation or inactivation of citramalate synthase enzyme activity can be achieved by the application of various methods well known in the art. Replacing a gene encoding the enzyme on a chromosome with a mutated gene such that, for example, the activity of the enzyme is completely removed, including when the activity of the enzyme is completely removed; Introducing a mutation into an expression control sequence (eg, a promoter) of a gene on a chromosome encoding said enzyme; A method of replacing an expression control sequence of a gene encoding the enzyme with a weak or inactive sequence; Deleting all or part of a gene on a chromosome that encodes the enzyme; Introducing an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to a transcript of a gene on the chromosome to inhibit translation from the mRNA to an enzyme; The method of artificially adding a sequence complementary to the SD sequence in front of the SD sequence of the gene encoding the enzyme to form a secondary structure to make the attachment of ribosomes impossible and the ORF (open reading frame) of the sequence Reverse transcription engineering (RTE) method for adding a promoter to reverse transcription at the 3 'end, and the like can be achieved by a combination thereof, but is not limited thereto.

According to one embodiment of the invention, the citramalate synthase may be one encoded by the leuA (2-isopropylmalate synthase) gene.

In the present invention, leuA (2-isopropylmalate synthase) gene has high homology with cimA (citramalate synthase) gene, and is known to play a role of citramalate synthase in Corynebacterium sp. According to one embodiment of the invention, the leuA gene consists of the nucleotide sequence of SEQ ID NO: 14.

According to one embodiment of the present invention, the weakened or inactivated activity of citramalate synthase is reduced expression of the leuA gene encoding citramalate synthase, thereby weakening or inactivating the activity of citramalate synthase. It may have been.

In the present invention, "reduced expression" means that the expression level of the gene is reduced from the original expression amount, and includes a case where expression is completely inhibited and no expression is made. In the case of reducing the expression in a microorganism, genetic initiation may be performed to replace the initiation codon of gene translation to reduce its expression or to reduce the expression level of an existing gene. The method of modifying a sequence for expression control may be performed by inducing a mutation in an expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in a nucleic acid sequence of the expression control sequence, or by promoting a promoter to a weaker promoter. It may be carried out by a method such as replacement. The expression control sequence includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosomal binding site, and a sequence that controls the termination of transcription and translation. If a gene that reduces expression is present in the microorganism to be mutated, for example, by replacing a native promoter that drives the expression of the gene with a weak promoter or introducing a mutation Can reduce gene expression. Furthermore, the expression of these genes can be reduced by deleting all or part of the existing genes.

According to one embodiment of the invention, the reduction of the expression of the leuA gene is carried out by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the nucleic acid sequence, or by replacing with a weaker promoter. Or the like.

Mutant strains of the genus Corynebacterium lacking the leuA gene according to an embodiment of the present invention is a by-product of citramalate production is reduced than the parent strain, and acetyl-CoA is saved to increase L-glutamic acid production Can be .

According to one embodiment of the present invention, the strain of the genus Corynebacterium comprises at least one leucine selected from the group consisting of leucine (93) and leucine (109), the amino acid sequence of the YggB protein represented by SEQ ID NO: 15. Mutations substituted with alanine or valine may be additionally induced.

In the present invention, the yggB gene refers to a gene encoding a mechanical sensory channel. The yggB gene is expressed in Genbank Accession No. It corresponds to the complementary sequence of the sequence of 1,336,092 to 1,337,693 among the genome sequences registered by NC_003450, and is also called NCgl1221. YggB protein encoded by the yggB gene, GenBank accession No. It is registered as NP_600492. According to one embodiment of the invention, the protein YggB yggB gene encrypted consists of the amino acid sequence of SEQ ID NO: 15.

According to one embodiment, the mutation in which one or more leucine selected from the group consisting of leucine (93) and 109 leucine in the amino acid sequence of the YggB protein represented by SEQ ID NO: 15 by alanine or valine is substituted for L- By removing the methyl group involved in the pore of the YggB protein from which glutamic acid can be removed, the pore diameter is increased, so that the strain having the mutation can improve the production capacity of L-glutamic acid.

Specifically, the variant strain may be transformed by a vector comprising a mutant of the YggB protein by a mutation or a polynucleotide encoding a mutant of the YggB protein.

In the present invention, "vector" refers to any medium for cloning and / or transferring bases to a host cell. The vector may be a replica that other DNA fragments can bind to and result in replication of the bound fragments. A "replicating unit" refers to any genetic unit (eg, plasmid, phage, cosmid, chromosome, virus) that functions as a self-unit of DNA replication in vivo, i.e., is replicable by its own regulation. . In the present invention, the vector is not particularly limited as long as it can be replicated in the host, and any vector known in the art may be used. The vectors used in the preparation of the recombinant vector may be plasmids, cosmids, viruses and bacteriophages in a natural state or in a recombinant state. For example, pWE15, M13, [lambda] EMBL3, [lambda] EMBL4, [lambda] FIXII, [lambda] DASHII, [lambda] ZAPII, [lambda] gt10, [lambda] gt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector. , pBluescript II system, pGEM system, pTZ system, pCL system and pET system can be used. The usable vector is not particularly limited and may be a known expression vector, but is not limited thereto.

In the present invention, "transformation" is to introduce a gene into the host cell to be expressed in the host cell, the transformed gene is located in the host cell chromosome insertion or chromosome if it can be expressed in the host cell Anything may be included without limitation.

The mutant strain of Corynebacterium, in which the leuA gene was deleted and the mutation in the YggB protein, according to one embodiment of the present invention, increased the production of L-glutamic acid and reduced the incubation time for L-glutamic acid production more efficiently than the parent strain. Can produce L-glutamic acid.

According to one embodiment of the present invention, the strain Corynebacterium may be Corynebacterium glutamicum ( Coynebacterium glutamicum ).

According to one embodiment of the invention, the strain Corynebacterium genus Corynebacterium ammonia genes ( Corynebacterium ammoniagenes ), Corynebacterium acetoacidophilum , Corynebacterium aceto glutamicum Corynebacterium acetoglutamicum , Corynebacterium alkanolyticum , Corynebacterium callunae , Corynebacterium lilium , Corynebacterium melasecola ), Corynebacterium thermo amino to Ness (Corynebacterium thermoaminogenes), Corynebacterium epi when Enschede (Corynebacterium efficiens), or Corynebacterium Herr cool-less (but Corynebacterium herculis the like), but is not limited to such.

According to one embodiment of the present invention, the strain Corynebacterium genus is most preferably Corynebacterium glutamicum KCTC 11558BP.

Another aspect of the invention

(a) culturing a strain of genus Corynebacterium in a medium; And

(b) recovering L-glutamic acid from the cultured mutant strain or culture medium.

According to one embodiment of the present invention, the culturing may be made according to suitable media and culture conditions known in the art, and those skilled in the art may easily adjust the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, but is not limited to, batch culture, continuous culture, fed-batch culture, or combination culture thereof.

According to one embodiment of the invention, the medium must meet the requirements of the particular strain in a suitable manner and can be appropriately modified by one skilled in the art. Culture media for strains of the genus Corynebacteria can be referred to, but are not limited to, the Manual of Methods for General Bacteriology. American Society for Bacteriology.Washington D.C., USA, 1981.

According to one embodiment of the present invention, the medium may include various carbon sources, nitrogen sources and trace element components. Carbon sources that may be used include, but are not limited to, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, Fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto. Nitrogen sources that may be used may include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used individually or as a mixture, but are not limited thereto. Sources of phosphorus that may be used may include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium may contain a metal salt such as magnesium sulfate or iron sulfate required for growth, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be included. Also suitable precursors to the culture medium can be used. The medium or individual components may be added batchwise or continuously in a suitable manner to the culture medium during the culturing process, but is not limited thereto.

According to one embodiment of the present invention, during the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microbial culture in a suitable manner to adjust the pH of the culture. In addition, antifoaming agents such as fatty acid polyglycol esters can be used during the culturing to suppress bubble formation. Additionally, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (eg, air) may be injected into the culture. The temperature of the culture may be usually 20 ℃ to 45 ℃, for example 25 ℃ to 40 ℃. The incubation period can continue until useful material is obtained at the desired yield, for example 10 to 160 hours.

According to one embodiment of the invention, the step of recovering L-glutamic acid in the cultured mutant strain and culture medium is collected or L-glutamic acid produced from the medium using a suitable method known in the art depending on the culture method It can be recovered. For example centrifugation, filtration, extraction, spraying, drying, steaming, precipitation, crystallization, electrophoresis, fractional dissolution (e.g. ammonium sulfate precipitation), chromatography (e.g. ion exchange, affinity, hydrophobicity and size exclusion) ) May be used, but is not limited thereto.

According to one embodiment of the present invention, the step of recovering glutamic acid may be carried out by centrifugation of the culture at low speed to remove the biomass and the obtained supernatant may be separated by ion exchange chromatography.

According to one embodiment of the invention, the step of recovering the L- glutamic acid may comprise the process of purifying L- glutamic acid.

Mutant strain according to an embodiment of the present invention is a weakened or inactivated activity of citramalate synthase to reduce the production of by-product citramalate, excellent production capacity of L- glutamic acid, additionally mutated YggB protein Since the strain enhances the production of glutamic acid to improve the yield of L-glutamic acid, it is possible to produce L-glutamic acid more effectively by using the mutant strain.

1 shows the synthetic route of citramalate.
Figure 2 shows a pKLA vector prepared for disrupting the leuA gene in Corynebacterium glutamicum KCTC 11558BP strain.
Figure 3 shows the positions of the primers (primers 1, 4) in the deletion region of the leuA gene.
4A shows the structure of the YggB protein.
4B shows the pore structure of the YggB protein in which the 93rd leucine is substituted with alanine in the amino acid sequence of the YggB protein.
4C shows the pore structure of the YggB protein in which the 109th leucine is substituted with alanine in the amino acid sequence of the YggB protein.
Figure 5 shows the pKY93 vector prepared to replace the 93rd amino acid in the amino acid sequence of the YggB protein with another amino acid in the I10 strain.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are provided to illustrate one or more embodiments by way of example, but the scope of the present invention is not limited to these examples.

Example 1 Intracellular Metabolite Analysis of Strain Fermentation Termination

Corynebacterium glutamicum KCTC 11558BP strain is a mutant strain obtained by treating nitrosoguanidine (NTG) which chemically causes DNA mutation in I6 strain, and has low molasses resistance as the main carbon source by having molasses resistance. Can be used to produce L-glutamic acid in high yield (Korean Patent Publication No. 10-2011-0034718).

The amount of L-glutamic acid and metabolite material inside cells of Corynebacterium glutamicum I6 (hereinafter referred to as I6) and Corynebacterium glutamicum KCTC 11558BP (hereinafter referred to as KCTC 11558BP) having glutamic acid production capacity were analyzed.

25 ml of KCTC 11558BP and I6 in CM liquid medium (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, 50% NaOH 100 l, pH 6.8, 1L) After inoculation to the shaking culture at 200 ℃ 200 ℃. When cell growth reached the exponential phase during the culture, cells were separated from the culture via rapid vacuum filtration (Durapore HV, 0.45 m; Millipore, Billerica, Mass.). After the cell-adsorbed filter was washed twice with 10 ml of cooling water, the reaction was stopped by soaking for 10 minutes in methanol containing 5M morpholine ethanesulfonic acid and 5M methionine sulfone. After the same amount of chloroform and 0.4 times the volume of water was mixed with the extract obtained from this, only an aqueous phase was applied to a spin column to remove protein contaminants. The filtered extract was analyzed for relative citramalate amounts using capillary electrophoresis mass spectrometry, and the results are shown in Table 1 below.

In addition, high performance liquid chromatography (HPLC; Agilent, USA) using a C18 column (Apollo C18, 5 μm, 150 mm 4.6 mm) was performed to analyze L-glutamic acid production. The results are shown in Table 1 below. KH ₂ PO ₄ 8.85g / L, Tetrabutyl ammonium hydroxide solution 1.652g / L, Acetonitrile 10ml / L, Sodium azide 12.5mg / L and pH 3.15 An analytical buffer (adjusted with phosphoric acid) was used by filtering with 0.45 mu m membrane-filter (HA).

As shown in Table 1, it was confirmed that the citramalate produced by KCTC 11558BP increased about three times compared to the control group I6.

I6 (control) KCTC 11558BP L-Glutamic Acid Production (g / L) 34 37.5 Citramalate (relative amount) 130.2 330.1

Example 2. leuA Preparation of Corynebacterium glutamicum Mutant Strains Deleting Genes

1 shows the synthetic route of citramalate. Citramalate is produced by the cimA (citramalate synthase) gene in Escherichia coli, but the cimA gene is absent in Corynebacterium glutamicum. However, the Corynebacterium glutamicum cimA gene and the sheet ramal high homology leuA gene rate synthase so known to act as a (citramalate synthase) leuA (2- isopropylmalate synthase) shredding the by-product of sheet ramal rate in In order to further increase the productivity of L-glutamic acid by reducing and saving acetyl-CoA, a substrate used to prepare L-glutamic acid. SEQ ID NO: 14 shows the nucleotide sequence of the leuA gene.

Example 2-1. leuA  Preparation of Gene Disruption Vectors

Corynebacterium glutamicum KCTC 11558BP with L-glutamic acid production was used for leuA gene (NCgl0245) disruption in Corynebacterium glutamicum.

 Chromosomal DNA of Corynebacterium glutamicum KCTC 11558BP strain was isolated and used as a template using primers 1 and 2 and primers 3 and 4, respectively, at 95 ° C. for 30 seconds at 57 ° C. PCR was performed by repeating 30 cycles under conditions of 30 seconds at 72 ° C. and 60 seconds at 72 ° C., and the obtained PCR products (H1 and H2 in FIG. 2) were purified.

The purified PCR product was again used as a template for crossover polymerase chain reaction (PCR) and amplified once again with primer sets 1 and 4.

The 1600 bp crossover PCR product for the deletion of the leuA gene was purified and digested with Xba I and Sal I restriction enzymes (Takara, Japan) and then with the same restriction enzyme pK19mobSacB vector (Gene, 145: 69-73, 1994). Was cloned to prepare a vector for leuA gene disruption, which was named pKLA. Figure 2 shows a pKLA vector prepared for disrupting the leuA gene in Corynebacterium glutamicum KCTC 11558BP strain.

Primer name Sequence (5'-3 ') SEQ ID NO: primer 1 5'-TCTAGACCCTTCCCGGAAACCCCCAC-3 ' SEQ ID NO: 1 primer 2 5'-GGGGTTTCGATCTTGGCAGG-3 ' SEQ ID NO: 2 primer 3 5'-ACCGCGCGCTGGACGTCAAC-3 ' SEQ ID NO: 3 primer 4 5'-GTCGACTGGCCGAAACGCTTAGCGCT-3 ' SEQ ID NO: 4

Example 2-2. Transformation of Corynebacterium glutamicum strains and leuA  Deletion Mutant Strain Preparation

Corynebacterium glutamicum KCTC 11558BP strain made available for transformation through electrocompetent cell preparation using the method of van der Rest (Appl. Microbiol. Biotechnol., 52, 541-545, 1999) KCTC 11558BP) was introduced into the vector pKLA prepared in Example 2-1 via electric shock.

Specifically, KCTC 11558BP strain was first cultured in 100 ml of 2YT medium (tryptone 16 g / l, yeast extract 10 g / l, sodium chloride 5 g / l) to which 2% glucose was added, and 1 mg / ml in the same medium except glucose. Concentrations of isonicicotinic acid hydrazine and 2.5% glycine were added. Then, after inoculating the seed culture solution so that the OD ₆₁₀ value is 0.3, the OD ₆₁₀ value was 1.2 to 1.4 by incubating at 18 ° C and 180 rpm for 12 to 16 hours. After standing in ice for 30 minutes, centrifuged at 4 ℃, 4000rpm for 15 minutes. The supernatant was then discarded and the precipitated KCTC 11558BP strain was washed four times with 10% glycerol solution and finally resuspended in 0.5 ml of 10% glycerol solution. Electroporation was performed using a Bio-Rad electroporator. After inserting the competent cell prepared by the above method into the electroporation cuvette (0.2 mm) and adding the pKLA vector prepared in Example 2-1, the electric shock under the conditions of 2.5 kV, 200 Ω and 12.5 ㎌ Was added. Immediately after the electric shock, 1 ml of Regeneration medium (Brain Heart infusion 18.5 g / l, Sorbitol 0.5 M) was added and heat-treated at 46 ° C. for 6 minutes. After cooling at room temperature, transfer to a 15 ml cap tube and incubate at 30 ° C. for 2 hours, followed by selection medium (tryptone 5 g / l, NaCl 5 g / l, yeast extract 2.5g / l, Brain Heart infusion powder 18.5 g / l). , 15 g / l of agar, 91 g / l of sorbitol, and 20 µg / l of kanamycine. Colonies generated by incubating at 30 ° C. for 72 hours were incubated in BHI medium until stationary phase to induce secondary recombination, diluted to 10 ^-5 to 10 ^-7 and plated on antibiotic-free plates (containing 10% sucrose) to kanamycin resistance. Strains were grown in medium without 10% sucrose. The selected strains were identified as Corynebacterium glutamicum strains in which the leuA gene was deleted using the primers 1 and 4 described in Table 2 above, and the deletion strains in which the leuA gene was deleted were designated as "I10." Figure 3 shows the positions of the primers 1, 4 in the deletion of the leuA gene.

Example 2-3. leuA Determination of Nutritional Needs of Deficient Strains

The leuA deficient strain (hereinafter, referred to as I10) prepared in Example 2-2 is a leucine nutritional strain because the leuA gene is deleted, and to confirm this, the leuA deficient strain was prepared in the minimum medium and leucine-added medium prepared in the composition of Table 3 below. Smear and incubate at 30 ℃ for 24 hours to measure the growth degree is shown in Table 4 the results.

Minimum medium composition table glucose 20 g / L MgSO ₄ . 7H ₂ O 0.5g / L FeSO ₄ . 7H ₂ O 20mg / L MnSO ₄ . 5H ₂ O 20mg / L urea 2.5g / L (NH ₄ ) ₂ SO ₄ 5g / L KH ₂ PO ₄ 1.5g / L K ₂ HPO ₄ 1.5g / L Thiamine-HCl 200 µg / L Biotin 200 µg / L Nicotinic acid 200 µg / L Vitamin B2 200 µg / L Cyanocobalamine 100 µg / L

As shown in Table 4, the growth of the I10 strain was drastically reduced compared to the parent strain Corynebacterium glutamicum KCTC 11558BP strain in the minimal medium without leucine and the growth was recovered by the addition of leucine. Therefore, the leuA gene defect was identified in the I10 strain.

KCTC 11558BP I10 Minimum badge ++++ + Minimum medium + 50 ppm leucine ++++ ++++

Example 3. leuA  Confirmation of L-glutamic acid and citramalate production of Corynebacterium glutamicum strain (I10)

The leuA deficient strain (hereinafter referred to as I10) and Corynebacterium glutamicum KCTC 11558BP (hereinafter referred to as KCTC 11558BP) were incubated at 30 ° C. for 24 hours using an active plate medium prepared at the content shown in Table 5 below. Thereafter, 10 ml of the L-glutamic acid flask medium prepared in the content shown in Table 6 was put in a 100 ml flask, and the incubated bacteria were inoculated in a loop and incubated at 30 ° C., 200 rpm, for 48 hours. Culture termination was used to perform L-glutamic acid production and intracellular metabolite analysis. L-glutamic acid production and relative citramalate amounts were performed in the same manner as described in Example 1.

Table 7 shows L-glutamic acid production and relative citramalate amounts in I10 and KCTC 11558BP. As shown in Table 7, it was confirmed that the production of L- glutamic acid in the I10 strain increased and citramalate significantly decreased.

Active Plate Badge glucose 5g / L Yeast extract 10 g / L urea 3g / L KH ₂ PO ₄ 1 g / L Biotin 2 µg / L Soybean Hydrolyzate 0.1% v / v Leucine 50 mg / L Agar 20 g / L pH 7.5

L-glutamic acid flask medium composition table glucose 70 g / L MgSO ₄ . 7H ₂ O 0.4g / L urea 2 g / L KH ₂ PO ₄ 1 g / L Soybean Hydrolyzate 1.5% v / v (NH ₄ ) ₂ SO ₄ 5g / L FeSO ₄ . 7H ₂ O 10mg / L MnSO ₄ . 5H ₂ O 10mg / L Thiamine-HCl 200 µg / L Biotin 2 µg / L Calcium carbonate 50 g / L pH 7.6

KCTC 11558BP I10 L-Glutamic Acid Production (g / L) 37.5 39.7 Citramalate (relative amount) 320.4 8.5

Example 4. Preparation of YggB Mutant Strains

As a result of metabolite analysis of leuA- deficient strain (I10), it was confirmed that many L-glutamic acid was accumulated in the cells, thereby enhancing the release of L-glutamic acid.

YggB is a mechanosensitive channel homolog known to play an important role in the release of L-glutamic acid from Corynebacterium glutamicum. SEQ ID NO: 15 shows amino acid sequence of YggB protein. 4A shows the structure of the YggB protein. The protein structure of YggB was analyzed to explore variations that could enhance emissions.

4B shows the pore structure of the YggB protein in which the 93rd leucine is substituted with alanine in the amino acid sequence of the YggB protein, and FIG. 4C shows the pore of the YggB protein in which the 109th leucine is substituted with alanine in the amino acid sequence of the YggB protein. pore) structure.

Example 4-1. Preparation of a Strain Substituting the 93rd Amino Acid in the Amino Acid Sequence of the YggB Protein

As shown in FIG. 4B, when the 93rd leucine is replaced with alanine in the amino acid sequence of the YggB protein in order to expand the L-glutamic acid migration pathway, the methyl group involved in the narrow part of the pore is removed to increase the pore diameter. Mutant strains were prepared by estimation.

Chromosomal DNA of the I10 strain prepared in Example 2-2 was isolated and used as a template, using primers 5,6 sets and primers 7,8 sets, respectively, at 95 ° C. for 30 seconds and 57 ° C. PCR was carried out by repeating 30 cycles under conditions of 30 seconds and 72 ° C. for 60 seconds, and the obtained PCR product was purified. The purified PCR product was used as a template and amplified by crossover PCR using primers 5,8. The amplified product was digested by sequencing and digested with Xba I and Sal I restriction enzymes (Takara, Japan). The pK19mobSacB vector was digested with the same restriction enzyme and ligated with the amplified crossover PCR product to construct a vector, which was named pKY93. Primer sequences used are listed in Table 8. Figure 5 shows the pKY93 vector for substitution of the 93rd amino acid in the amino acid sequence of the YggB protein in the I10 strain.

primer Sequence (5'-3 ') SEQ ID NO: primer 5 5'-TCTAGATTGAGAAGCTGCCACATTCAC-3 ' SEQ ID NO: 5 primer 6 5'-GCAGCGCCCGCGGCAGAGAAACCAAAAGCC-3 ' SEQ ID NO: 6 primer 7 5'-CTCTGCCGCGGGCGCTGCGATTCCGGCAAC-3 ' SEQ ID NO: 7 primer 8 5'-GTCGACGATCTGGTTTTGCTGTTTCTTCCGG-3 ' SEQ ID NO: 8 primer 9 5'-GACTGCGCACCAGCACCAATGGCAGCTGAC-3 ' SEQ ID NO: 9 primer 10 5'-GGTGCTGGTGCGCAGTCGATTGTTGCGGAC-3 ' SEQ ID NO: 10 primer 11 5'-TCTAGAGGAATCAGGATTCTCACAAAGTTCAGGC-3 ' SEQ ID NO: 11 primer 12 5'-CGGAATCGCAGTGCCCGCGAGAGAGAAACC-3 ' SEQ ID NO: 12 primer 13 5'-CGCGGGCACTGCGATTCCGGCAACCATTGC-3 ' SEQ ID NO: 13

In the same manner as in Example 2-2, the strain transformed by introducing the pKY93 vector into the I10 strain was selected and named as I10-93. In the I10-93 variant, leucine, the 93rd amino acid of the YggB protein, was substituted with alanine.

Example 4-2. Construction of a Strain Substituted with the 109th Amino Acid in the Amino Acid Sequence of the YggB Protein

As shown in FIG. 4C, when the 109th leucine is replaced with alanine in the amino acid sequence of the YggB protein to expand the L-glutamic acid migration pathway, the methyl group involved in the narrow portion of the pore is removed, thereby increasing the pore diameter. Mutant strains were prepared by estimation.

Chromosomal DNA of I10 strain prepared in Example 2-2 was isolated and used as a template, using primers 5, 9 sets and primers 10, 8 sets, respectively, at 95 ° C. for 30 seconds, at 57 ° C. PCR was carried out by repeating 30 cycles under conditions of 30 seconds and 72 ° C. for 60 seconds, and the obtained PCR product was purified. The purified PCR product was used as a template and amplified by crossover PCR using primers 5 and 8. The amplified product was digested by sequencing and digested with Xba I and Sal I restriction enzymes (Takara, Japan). The pK19mobSacB vector was digested with the same restriction enzyme and ligated with the amplified crossover PCR product to construct a vector, which was named pKY109. Primer sequences used are listed in Table 8.

In the same manner as in Example 2-2, the strain transformed by introducing the pKY109 vector into the I10 strain was selected and named as I10-109. In the I10-109 variant strain, leucine, the 109th amino acid of the YggB protein, was substituted with alanine.

Example 5 L-Glutamic Acid Productivity of Mutant Strains

In the same manner as in Example 1 and Example 3, the amount and time required for the production of L-glutamic acid by the strains I10, I10-93 and I10-109 at the flask scale were measured and the results are shown in Table 9.

I10 I10-93 I10-109 L-Glutamic Acid Production (g / L) 39 42.5 45.2 Hours (hr) 48 48 44

As shown in Table 9, L-glutamic acid production was higher than that of the I10 strain in the I10-93 and I10-109 variant strains that induced pore expansion of the YggB protein. In particular, in the case of I10-109 strain L- glutamic acid production significantly increased than I10 and the fermentation time is also shortened from 48 hours to 44 hours, it is possible to efficiently produce L- glutamic acid.

<110> DAESANG CORPORATION <120> Mutant strain with enhanced L-glutamic acid productivity and          method for preparing L-glutamic acid using the same <130> PN180417 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 1 tctagaccct tcccggaaac ccccac 26 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 2 ggggtttcga tcttggcagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 3 accgcgcgct ggacgtcaac 20 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 4 <400> 4 gtcgactggc cgaaacgctt agcgct 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 5 <400> 5 tctagattga gaagctgcca cattcac 27 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 6 <400> 6 gcagcgcccg cggcagagaa accaaaagcc 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 7 <400> 7 ctctgccgcg ggcgctgcga ttccggcaac 30 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer 8 <400> 8 gtcgacgatc tggttttgct gtttcttccg g 31 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 9 <400> 9 gactgcgcac cagcaccaat ggcagctgac 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 10 <400> 10 ggtgctggtg cgcagtcgat tgttgcggac 30 <210> 11 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer 11 <400> 11 tctagaggaa tcaggattct cacaaagttc aggc 34 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 12 <400> 12 cggaatcgca gtgcccgcga gagagaaacc 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer 13 <400> 13 cgcgggcact gcgattccgg caaccattgc 30 <210> 14 <211> 1851 <212> DNA <213> Corynebacterium sp. <400> 14 atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct 60 cgcaacgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc 120 tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat 180 aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct 240 ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg 300 ggcttcaaag aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt 360 cgtgagatca tcgaaaaggg catgatccct gacgatgtca ccattcaggt tctggttcag 420 gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc 480 gtgcacttct acaactccac ctccatcctg cagcgcaacg tggtgttccg catggacaag 540 gtgcaggtga agaagctggc taccgatgcc gctgaactaa tcaagaccat cgctcaggat 600 tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt 660 gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac 720 ccaatgatca tcaacctgcc ttccaccgtt gagatgatca cccctaacgt ttacgcagac 780 tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac 840 ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct 900 gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc 960 accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata 1020 cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca 1080 tacggcggtg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag 1140 ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg 1200 gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc 1260 ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct 1320 tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc 1380 accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg 1440 gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc 1500 gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac 1560 ggcaaggacg tcaccgtcga tggccgcggc aacggcccac tggccgctta cgccaacgcg 1620 ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg 1680 ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg 1740 ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta 1800 aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a 1851 <210> 15 <211> 533 <212> PRT <213> Corynebacterium sp. <400> 15 Met Ile Leu Gly Val Pro Ile Gln Tyr Leu Leu Tyr Ser Leu Trp Asn   1 5 10 15 Trp Ile Val Asp Thr Gly Phe Asp Val Ala Ile Ile Leu Val Leu Ala              20 25 30 Phe Leu Ile Pro Arg Ile Gly Arg Leu Ala Met Arg Ile Ile Lys Arg          35 40 45 Arg Val Glu Ser Ala Ala Asp Ala Asp Thr Thr Lys Asn Gln Leu Ala      50 55 60 Phe Ala Gly Val Gly Val Tyr Ile Ala Gln Ile Val Ala Phe Phe Met  65 70 75 80 Leu Ala Val Ser Ala Met Gln Ala Phe Gly Phe Ser Leu Ala Gly Ala                  85 90 95 Ala Ile Pro Ala Thr Ile Ala Ser Ala Ala Ile Gly Leu Gly Ala Gln             100 105 110 Ser Ile Val Ala Asp Phe Leu Ala Gly Phe Phe Ile Leu Thr Glu Lys         115 120 125 Gln Phe Gly Val Gly Asp Trp Val Arg Phe Glu Gly Asn Gly Ile Val     130 135 140 Val Glu Gly Thr Val Ile Glu Ile Thr Met Arg Ala Thr Lys Ile Arg 145 150 155 160 Thr Ile Ala Gln Glu Thr Val Ile Ile Pro Asn Ser Thr Ala Lys Val                 165 170 175 Cys Ile Asn Asn Ser Asn Asn Trp Ser Arg Ala Val Val Val Ile Pro             180 185 190 Ile Pro Met Leu Gly Ser Glu Asn Ile Thr Asp Val Ile Ala Arg Ser         195 200 205 Glu Ala Ala Thr Arg Arg Ala Leu Gly Gln Glu Lys Ile Ala Pro Glu     210 215 220 Ile Leu Gly Glu Leu Asp Val His Pro Ala Thr Glu Val Thr Pro Pro 225 230 235 240 Thr Val Val Gly Met Pro Trp Met Val Thr Met Arg Phe Leu Val Gln                 245 250 255 Val Thr Ala Gly Asn Gln Trp Leu Val Glu Arg Ala Ile Arg Thr Glu             260 265 270 Ile Ile Ser Glu Phe Trp Glu Glu Tyr Gly Ser Ala Thr Thr Thr Ser         275 280 285 Gly Thr Leu Ile Asp Ser Leu His Val Glu His Glu Glu Pro Lys Thr     290 295 300 Ser Leu Ile Asp Ala Ser Pro Gln Ala Leu Lys Glu Pro Lys Pro Glu 305 310 315 320 Ala Ala Ala Thr Val Ala Ser Leu Ala Ala Ser Ser Asn Asp Asp Ala                 325 330 335 Asp Asn Ala Asp Ala Ser Val Ile Asn Ala Gly Asn Pro Glu Lys Glu             340 345 350 Leu Asp Ser Asp Val Leu Glu Gln Glu Leu Ser Ser Glu Glu Pro Glu         355 360 365 Glu Thr Ala Lys Pro Asp His Ser Leu Arg Gly Phe Phe Arg Thr Asp     370 375 380 Tyr Tyr Pro Asn Arg Trp Gln Lys Ile Leu Ser Phe Gly Gly Arg Val 385 390 395 400 Arg Met Ser Thr Ser Leu Leu Leu Gly Ala Leu Leu Leu Leu Ser Leu                 405 410 415 Phe Lys Val Met Thr Val Glu Pro Ser Glu Asn Trp Gln Asn Ser Ser             420 425 430 Gly Trp Leu Ser Pro Ser Thr Ala Thr Ser Thr Ala Val Thr Thr Ser         435 440 445 Glu Thr Ser Ala Pro Val Ser Thr Pro Ser Met Thr Val Pro Thr Thr     450 455 460 Val Glu Glu Thr Pro Thr Met Glu Ser Asn Val Glu Thr Gln Gln Glu 465 470 475 480 Thr Ser Thr Pro Ala Thr Ala Thr Pro Gln Arg Ala Asp Thr Ile Glu                 485 490 495 Pro Thr Glu Glu Ala Thr Ser Gln Glu Glu Thr Thr Ala Ser Gln Thr             500 505 510 Gln Ser Pro Ala Val Glu Ala Pro Thr Ala Val Gln Glu Thr Val Ala         515 520 525 Pro Thr Ser Thr Pro     530

Claims (5)

Corynebacterium sp. Mutant strain with weakened or inactivated citramalate synthase activity and improved L-glutamic acid production.
The variant strain of genus Corynebacterium according to claim 1, wherein the citramalate synthase is encoded by a leuA (2-isopropylmalate synthase) gene.
The mutant strain of Corynebacterium genus according to claim 1, wherein at least one leucine selected from the group consisting of leucine (93th amino acid) and leucine (109th amino acid) in the amino acid sequence of the YggB protein represented by SEQ ID NO: 15 is alanine or valine. A variant strain of Corynebacterium that is caused by further substitution.
The strain according to any one of claims 1 to 3, wherein the strain is Corynebacterium glutamicum .
(a) culturing the strain of Corynebacterium genus according to any one of claims 1 to 3 in a medium; And
(b) recovering L-glutamic acid in the cultured mutant strain or culture medium.

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